1. Field of the Invention
The invention relates to a process for sexing cow embryos and in particular,to a process for sexing cow embryos by method of polymerase chain reaction(PCR) against the amelogenin (bAML) genes located on both X- and Y- chromosomes of the Holstein dairy cattle. The invention further relates to a group of primers useful in such a process for sexing cow embryos and, specificaly, to a pair of sex-specific primers designed for amplify a single fragment of 467-bp from the female cattle and two fragments of 467-bp and 341-bp from the male one , respectively. In addition, the invention also provide a kit for sexing cow embryos.
2. Description of the Prior Art
Recently, as various technigues of animal reproduction are extensively advanced, animal husbandry is not restricted within traditional breeding tasks any more. Among these, the most promising ones are techniques related with embryo transplantation, such as embryo freezing, embryo cloning, embryo sexing and the like; since the intimately combined use of these techniques with embryo transplantation will promote managing efficiencies of animal husbandry, and thereby yields revolutionary benefits.
Several methods can be used for sexing animal embryos at present, including: karyotyping, H-Y antigen analysis, X-chromosome-linked enzymatic activity test, Y-specific probe hybridization, polymerase chain reaction method and the like (Bondioli el al., Theriogenology 31: 95-104, 1989).
The karyotyping method is relied principally on cytogenetic analysis against blastomeres of a embryo and determines sex type of the embryo on basis of the occuring of X or Y sex chromosomes. The karyotyping method had been developed since 1968, Gardner and Edwards (Nature 218: 346-348 ) observed directly blaetocysts of rabbits and determines the feminality of that rabbit embryo based on the fact that there is an inactivated X chromosome, or so-called Barr's body, existed in that cell, however, according to King (J. Reprod. Fert. 98: 335-340, 1984 ), this method is not suitable for sexing embryos of common domestic animals, since, in somatic cells of common domestic animals, observable Barr's body is less than 50%, and further, cytoplasm of embryo of common domestic animals including embryos of pork, beef, sheep and goat, are not as clear as rabbit embryo such that observation of Barr's body in those embryos is difficult to carry out. At present, karyotyping done on embryos of domestic animals consists usually of treating embryo firstly with chochicine to induce its blastomere in metaphase of cell division and verifying whether there is Y chromosome in the karyotype of the embryo according to the shape of the chromosome. However, sexing embryo based on karyotyping involving treatment with chochicine is subject to have several disadvantages in that its procedure is cumbersome and time consumptive as well as the identifiability of cell is low, and in particular, with single blastomere, the probability to obtain an identifiable karyotype is even smaller.
The X-linked enzymatic activity test is based on the fact that both X chromosomes of normal female mammal have function before the blastula stage in the development of the embryo, while one of the two X chromosomes will be inactivated after the blastula stage (Lyon, Biol. Rev. 47: 1-35, 1972 ); now, it is known that there are several enzymatic genes on X chromosome, such as, glucose-6-phosphate dehydrogenase (G6PD) and hypoxanthine phosphoribosyl transferase (HPRT), so that the number of X chromosomes present in an embryo can be predicted by comparing strengths of activities of these enzymes. Although damage due to microscopic operation can be avoided during application of such enzymatic activity test on sexing of embryo, the survival rate of the embryo is low owing to the direct exposure of embryo in a reaction solution (Williams, Theriogenology 25:733-739, 1986),and furthermore, X-inactivation stages have not been verified among various species yet, which lead this method not being widely used.
Sex-determination of embryos by means of Y-specific nucleotides is based on two molecular biological principles: the first one comprising of using Y-specific DNA sequence as probe to hybridize genomic DNA extracts derived from the embryo; while the second one comprising of designing suitable primers based on Y-specific nucleotide sequences and carrying out in vitro amplification of gene sequence by means of PCR. Wherein, because of their high sensitivity, high accuracy and high efficiency, polymerase chain reaction method will become the most potentially practical technique among various methods.
In order to apply PCR on sex-determination of embryo of domestic animals, a nucleotide sequence specific against sex should be recognized at first, such as SRY one associated with testis determining factor (Sinclair el al., Nature 346:240-244, 990), certain Y-specific repetitive sequences (Nakahori et al., Nucleic Acid Res. 14:7569-7580, 1986), ZFX, ZFY genes homologous to X and Y chromosomes (zinc finger X chromosome, zinc finger Y chromosome)(Pollevick el al.,Bio/Technol. 10:805-807, 1989) and so on which are known to have sex-specific NUCLEOTIDE sequences. However, in those PCR-bassed sexing methods described above, each primer pair can recognize sex-specific gene fragments derived from only one sex chromosome whereas it can not detect simultaneously fragments derived from both of X and Y chromosomes so that an internal control primer derived from intrinsic gene must be added in the reaction. Since the existence of two groups of primer in the enzymatic amplification may readily result in competition therebetween and increase the probability of formation of primer dimer during in vitro amplification of gene, the simplicity of the method is greatly reduced consequently. Against this defect, the invention designs a group of primers homologous to both of X and Y chromosomes, which can amplify gene fragments of different lengths simultaneously from X chromosome and from Y chromosome, so that sex type of early cow embryo before implantation can be determined straightwardly from results of electrophoresis.